Engineered enzymes and their use for synthesis of thioglycosides

ABSTRACT

Mutant glycosidases in which the amino acid in the active site that serves as the acid, base or acid/base-catalyst is converted from a carboxylic acid to some other amino acid (for example to a simple alkyl, as in alanine or glycine) can catalyze the reaction of a thiosugar acceptor and an activated donor to form a thioglycoside. The “thioglycoligases” represent a novel class of mutant enzymes, and represent a first aspect of the invention. Thioglycoligases can be used in accordance with the method of the invention to couple a thiosugar acceptor and an activated donor to form a thioglycoside. By selection of the donor and acceptor species, as well as the specific enzyme employed, thioglycosides of different structure and stereochemistry can be obtained.

This application claims the benefit of U.S. Provisional Application No.60/410,502, filed Sep. 12, 2002, which application is incorporatedherein by reference in those jurisdictions permitting suchincorporation.

BACKGROUND OF THE INVENTION

This application relates to engineered enzymes, and to their use for thesynthesis of thioglycosides.

Carbohydrate mimetics that are resistant towards enzymatic hydrolysishave proven to be useful as competitive glycosidase inhibitors andtherefore have potential as therapeutics. Thioglycosides, in which theglycosidic oxygen has been replaced by sulfur, have been especiallyvaluable as stable glycoside analogues in a range of studies ofglycosidases, both as competitive inhibitors, e.g. forα-L-fucosidases,^([1]) pancreatic α-amylase^([2)] or forcellulases,^([3]) and recently in the formation of stable complexes forX-ray crystallography analysis, e.g. with endoglucanase Cel7B fromFusarium oxysporum, ^([4]) maize β-glucosidase ZMGlu1,^([5]) barleyβ-D-glucan glucohydrolase,^([6]) endoglucanase Cel5A^([7]) or E. colimaltodextrin phosphorylase (MalP),^([8]) and they are gaining increasinginterest as targets for the pharmaceutical industry.^([9])

The chemical synthesis of thioglycosides has been achieved viaglycosylation of thioacceptors with activated glycosyldonors,^([10], [11], [12]) via the S_(N)2 reaction of 1-thio sugars withactivated acceptors^([13]) and via Michael addition of 1-thiosugars toα,β-unsaturated systems;^([14]) all routes involve numerous protectionand deprotection steps and require good control of anomericstereochemistry.

The few naturally occurring thioglycosides belong to the family ofglucosinolates and are found in cruciferous plants.^([15]) The enzymesthat catalyze the formation of the thioglycosidic linkages,S-glucosyltransferases, have been purified from plant extracts,^([16])as well as cloned into and expressed from E. coli. ^([17]) However, nouseful enzymatic syntheses of thioglycosidic linkages in vitro have beenreported. Such approaches would be valuable.

The action of retaining glycosidases on glycosides is in most instancesmediated by two key active site amino acid residues, the catalyticnucleophile and the catalytic acid/base. We have previously reported onthe efficient synthesis of O-glycosidic linkages in oligosaccharides bythe use of retaining glycosidases that lack the catalytic nucleophile(glycosynthases), in conjunction with activated donors of the oppositeanomeric configuration of the natural substrate.^([18], [19], [20])

In order to facilitate the synthesis of thioglycosides of diversestructure, it would be useful to have an enzymatic methodology. It is anobject of the present invention to provide such methods for makingthioglycosides.

SUMMARY OF THE INVENTION

It has now been surprisingly found that mutant glycosidases in which theamino acid in the active site that serves as the acid, base oracid/base-catalyst is converted from a carboxylic acid to some otheramino acid (for example to a simple alkyl as in alanine or glycine) cancatalyze the reaction of a thiosugar acceptor and an activated donor toform a thioglycoside. The “thioglycoligases” represent a novel class ofmutant enzymes, and represent a first aspect of the invention.Thioglycoligases can be used in accordance with the method of theinvention to couple a thiosugar acceptor and an activated donor to forma thioglycoside. By selection of the donor and acceptor species, as wellas the specific enzyme employed, thioglycosides of different structureand stereochemistry can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the hydrolysis of a disaccharide within the active site ofa normal glycosidase enzyme which retains stereochemical configurationduring hydrolysis.

FIG. 2 shows the hydrolysis of a disaccharide within the active site ofa normal glycosidase enzyme which inverts stereochemical configurationduring hydrolysis.

FIG. 3 shows HPLC chromatograms of the Man2A E429A catalyzed reaction ofthe two competing acceptors pNP-Xyl (5.5 mM) and its thio analogue 6(5.5 mM) with varying concentrations of DNP-Man: a) 3.75 mM, b) 22 mM.Arrows indicate the elution of the O-linked disaccharides, 10 is theS-linked disaccharide.

FIG. 4 shows the mechanism of a retaining glycosidase: Glycosylation anddeglycosylation with wild type A) and acid/base mutant B).DNP=dinitrophenyl, Nu=nucleophile.

FIG. 5 shows enzymatic synthesis of thioglycosides 7 and 9.DNP=dinitrophenyl, pNP=para-nitrophenyl

FIG. 6 shows enzymatic synthesis of thioglycosides 8 and 10.DNP=dinitrophenyl, pNP=para-nitrophenyl.

FIG. 7 shows results of kinetic analysis on Abg E171A with 2,4-DNPβ-D-glucopyranoside (125 mM) as donor and with pNpβ-D-4-deoxy-4-thio-glucopyranoside as acceptor.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel method for the synthesis ofS-glycosidic linkages in oligosaccharides by use of glycosidases thatlack the catalytic acid/base amino acid residue as a result of amutation. Glycosidase enzymes can be classified as being either“retainers” because they retain the stereochemistry of the bond beingbroken during hydrolysis, or “inverters” because they invert thestereochemistry of the bond being broken during hydrolysis. The mutantenzymes of the present invention, which may be referred to herein asthioglycoligases, can be formed by mutation of retaining or inverting,alpha or beta glycosidases.

Normal stereochemistry retaining glycosidase enzymes have two carboxylicacid groups in the active site of the enzyme as shown generally inFIG. 1. One of these groups functions as an acid/base catalyst (labeledas group 1 in FIG. 1) and the other as a nucleophile (group 2 in FIG.1). The nucleophile group 2 forms a glycosyl-enzyme intermediate whichis then cleaved by water or some other nucleophile with the help of theacid/base catalyst group 1 to result in a hydrolyzed glycoside in whichthe stereochemistry has been maintained.

Normal stereochemistry inverting enzymes also have two carboxylic acidgroups in the active site of the enzyme as shown generally in FIG. 2. Ininverting enzymes, however, one of these groups functions as an acidcatalyst (labeled as group 3 in FIG. 2) and the other as a base catalyst(group 4 in FIG. 2). The acid catalyst group 3 protonates the acetaloxygen of the glycosyl donor molecule, making it a good leaving group,at the same time that the base catalyst group 4 deprotonates an acceptormolecule (water or HOR) allowing it to replace the leaving group withinversion of stereochemistry.

The present invention provides mutant forms of both retaining andinverting enzymes in which one of the two carboxylic acid amino acids inthe active site has been replaced with a different amino acid. Suchmutations provide enzymes which are effective to catalyze the formationof thioglycosides.

Enzymes to which the methodology of the present invention may beemployed include, for example, β-glucosidases, β-galactosidases,β-mannosidases, β-N-acetyl glucosaminidases, β-N-acetylgalactosaminidases, β-xylosidases, β-fucosidases, cellulases, xylanases,galactanases, mannanases, hemicellulases, amylases, glucoamylases,α-glucosidases, α-galactosidases, α-mannosidases, α-N-acetylglucosaminidases, α-N-acetyl galactosaminidases, α-xylosidases,α-fucosidases, neuraminidases/sialidases such as those from:Agrobacterium sp., Bacillus sp., Caldocellum sp., Clostridium sp.,Escherichia coli, Kluveromyces sp., Klebsiella sp., Lactobacillus sp.,Aspergillus sp., Staphylococcus sp., Lactobacillus sp., Butyrovibriosp., Ruminococcus sp., Sulfolobus sp., Scihizophyllum sp., Trichodermasp., Cellulomonas sp., Erwinia sp., Humicola sp., Pseudomonas sp.,Thermoascus sp., Phaseolus sp., Persea sp., Fibrobacter sp.,Phanaerochaete sp., Microbispora sp., Saccharomyces sp., Hordeumvulgare, Glycine max, Saccharomycopsis sp., Rhizopus sp., Nicotiana,Phaseolus sp., rat, mouse, rabbit, cow, pig, and human sources.Preferred enzymes in accordance with the invention are mutant forms ofretaining glycosidase enzymes.

In the enzymes of the present invention, one of the two amino acidresidues with the active carboxylic acid side chains is changed to adifferent amino acid which does not act as an acid/base catalyst (in thecase of a retaining enzyme) or as an acid catalyst (in the case of aninverting enzyme). Thus, in general, the substitution will involvereplacing the glutamic acid or aspartic acid residue of the wild-typeenzyme with alanine, glycine, valine, leucine, isoleucine, serine,threonine, cysteine, methionine, asparagine, glutamine, histidine,proline, phenylalanine, or tyrosine. Preferably, the substituted aminoacid will have a side chain of approximately equal or smaller size tothe side chain of the wild-type amino acid residue to avoid significantchanges to the size and shape of the active site.

Formation of a mutant enzyme with the mutation at the designated aminoacid in the active site is suitably performed using site directedmutagenesis to arrive at the desired result. In general, this involvesthe construction of a plasmid containing the coding sequence for thewild-type gene, and isolation of single stranded DNA. Copies are thenmade of the isolated plasmid DNA using a template dependant DNApolymerase and a primer which overlaps the site of the desired mutationand which differs from the wild-type sequence in the manner necessary toyield the desired mutation. The mutated plasmid is then transformed intoa host organism, e.g., E. coli. Transformants are initially selectedusing a marker contained within the plasmid, and then further selectedby sequencing of the expressed glycosidase enzyme to confirm the natureof the mutation.

Mutant enzymes according to the invention may be purified from thegrowth medium of the host organism by column chromatography, for exampleon DEAE-cellulose if desired.

Glycosidases of use in the invention are based upon wild-type enzymeswhich can be categorized as either alpha or beta glycosidases based uponthe anomeric configuration of the natural substrate. When the enzymemodified to form the thioglycoligase used is a retaining enzyme, theresulting thioglycoligase should be used with a donor and acceptorhaving the same anomeric configuration as each other (for example alphaacceptor with alpha donor) and the same anomeric configuration as thenatural substrate. When the enzyme modified to form the thioglycoligaseused is a retaining enzyme, the resulting thioglycoligase should be usedwith an acceptor having the same configuration as the natural substrateand with a donor of opposite configuration.

In accordance with the method of the invention, thioglycosides of thegeneral structure A-S-B, where A and B are both sugar moieties and areformed by the enzymatic coupling of A-X and HS-B using a mutantglycosidase enzyme, wherein X is an appropriate leaving group. Inaddition to consideration of stereochemistry based on the enzyme beingemployed, the donors and acceptors are selected based on the desiredproduct, i.e., a glucoside where a glucoside is a desired component, amannoside where a mannose is a desired component, etc. The specificdonor and acceptor are also suitably chosen in light of the doubledisplacement mechanism of retaining glycosidases first proposed byKoshland^([21]).

Specific examples of suitable donors include, without limitation,2,4-dinitrophenyl β-D-glucopyranoside (DNP-Glc); 2,5-dinitrophenylβ-D-mannopyranoside (DNP-Man); DNP β-cellobioside, pNP4′-deoxy-4′-thio-β-cellobioside and β-D-glucosyl azide. Specificexamples of suitable acceptors include, without limitationpara-nitrophenyl 4-deoxy-4-thio-β-D-glucopyranoside, para-nitrophenyl4-deoxy-4thio-β-D-galactopyranoside; methylumbelliferyl4-deoxy-4-thio-β-D-glucopyranoside, 4′-deoxy-4′-thio-cellobiose, pNP4′-deoxy-4′-thio-β-cellobioside, and pNPβ-D4-deoxy-4-thio-glucopyranoside.

(Scheme 1, FIG. 4): In the glycosylation step the concerted action ofthe catalytic acid/base (protonated, acting as an acid) and thecatalytic nucleophile (deprotonated) leads to the departure of theaglycon group and to the formation of the covalent glycosyl-enzymeintermediate (A). Use of a mutant glycosidase in which the catalyticacid/base residue has been replaced by a non catalytically activeresidue necessitates glycosyl donors with good leaving groups that donot need acid catalysis, e.g. dinitrophenyl groups, to allow formationof the glycosyl-enzyme intermediate (B). However, turnover of thatintermediate via transglycosylation to an oxygen acceptor isimpractically slow, since general base catalysis is required to speedthis step (A). The acid/base mutant therefore requires strongnucleophiles as acceptors that do not need general base catalysis (B).As a practical consequence, the method of the present invention makesuse of a thiosugar acceptor with a free thiol, for example, apara-nitrophenyl 4-deoxy-4-thio glycoside or 3′-deoxy-3′-thio lactose.The thiosugar may be a monosaccharide, an oligosaccharide, or apolysaccharide, and may itself contain thioglycoside linkages as aresult of repeated cycles of the method of the invention. The donorspecies is one having a good leaving group. Typically, such a “goodleaving group” will have a leaving group ability from an acetal centerequal to or greater than that of p-nitrophenol.

Earlier experiments revealed that small anionic molecules, such as N₃ ⁻,AcO⁻, HCO₂ ^(−[22]) and F⁻,^([23]) rescue the reaction by enhancing therate of the deglycosylation step. In the present study we exploit thisconcept to allow the synthesis of thiooligosaccharides by use ofdeoxythio sugars as nucleophilic acceptors that do not need basecatalysis in the deglycosylation step. In summary, our approach requiresactivated donor glycosides, such as dinitrophenyl glycosides, andchemically synthesized deoxythio sugars as acceptors in conjunction withmutant glycosidases modified at the acid/base position.

We have probed our strategy using the alanine acid/base mutants of tworetaining β-glycosidases, the β-glucosidase from Agrobacterium sp. AbgE171A and the β-mannosidase from Cellulomonas fimi Man2A E429A. Themutant Abg E171A was generated by site-directed mutagenesis by a‘megaprimer’ PCR method using three oligonucleotide primers, onecontaining the mutation^([19]) The purified product of the first PCRreaction served as a megaprimer in the second PCR reaction. The purifiedgene was subcloned into an expression vector, and after expression theprotein was purified in a single step by Ni²⁺-chelation chromatography.The mutant Man2A E429A was the same protein sample described by Zechelet al.^([23]).

As donors we chose the readily available dinitrophenyl glycosides2,4-dinitrophenyl β-D-glucopyranoside (DNP-Glc) for studies with themutant glucosidase and 2,5-dinitrophenyl β-D-mannopyranoside (DNP-Man)for experiments involving the mutant mannosidase.^([23]) The low pK_(a)values of 3.96 for 2,4-dinitrophenol and 5.15 for 2,5-dinitrophenolrender acid catalysis for the glycosylation step unnecessary. As anacceptor we initially chose para-nitrophenyl4-deoxy-4-thio-β-D-glucopyranoside (3) since the parent sugarpara-nitrophenyl β-D-glucopyranoside (PNP-Glc) acts as an excellentacceptor for both wild type and nucleophile mutant forms of Abg^([18])and Man2^(A) ^([20]) undergoing transfer preferentially to the4-hydroxyl. The chemical synthesis of the deoxythio sugar 3 was readilyachieved via regioselective protection of the galacto sugar, activationof the unprotected axial alcohol by formation of the triflate,nucleophilic substitution with thioacetate with inversion ofconfiguration and finally Zemplen deprotection while excluding oxygen(in the presence of DTT). Anaerobic conditions, even during theenzymatic reactions, are compulsory to prevent oxidation of the thiolsto disulfides.

Upon incubation of 3 (20 mM) with the appropriate donor (45 mM) andmutant enzyme (˜1 mg/ml) in phosphate buffered solutions at pH 6.8 TLCanalysis revealed that a new product with the expected mobility of adisaccharide was formed quite rapidly in each case. These products werestable towards hydrolysis by both the wild type and mutant enzymes. Bycontrast, incubation of the wild type enzymes with the appropriate DNPsugar donors and thiosugar acceptor 3 resulted in no significantformation of disaccharide. A novel enzymatic coupling reaction wastherefore occurring involving specific thiolinkage formation. This wasconfirmed by isolation of the products of the enzymatic reactions viasilica gel chromatography after acetylation. ¹H and ¹³C NMR analysis aswell as mass spectrometry revealed that the products formed were indeedsulfur linked disaccharides (Scheme 2, FIG. 5).

These extremely encouraging results led us to question whether theregiochemical outcome of the transglycosylation reaction could becontrolled by virtue of the location of the thiol within the acceptorsugar. We therefore synthesized and tested para-nitrophenyl4-deoxy-4-thio-β-D-xylopyranoside (6) as an acceptor for the twoenzymes. Previous studies with both the wild type Abg and itsnucleophile mutant had revealed that para-nitrophenyl β-D-xylopyranoside(PNP-Xyl) is an excellent acceptor substrate, but that transfer,surprisingly, occurred exclusively to the 3-position.^([18]) Use of 6 asan acceptor would reveal whether the greater nucleophilicity of thethiol controls the reaction outcome. Indeed, incubation of 6 with AbgE171A and DNP-Glc resulted in an excellent yield (79%) of thesulfur-linked disaccharide, as revealed by ¹H and ¹³C NMR analysis aswell as ESI-MS. Similarly, incubation of 6 with Man2A E429A and DNP-Manproduced, in 82% yield, the β(1-4) linked thiodisaccharide product(Scheme 3).

Interestingly, control reactions in which the parent sugarspara-nitrophenyl β-D-xylopyranoside (pNP-Xyl) and para-nitrophenylβ-D-glucopyranoside (pNP-Glc) were used as acceptors for Man2A E429A inthe presence of DNP-Man, showed the formation of disaccharide products,but only very slowly (However, no formation of disaccharides wasobserved when using pNP-Glc or pNP-Xyl with Abg E171A in the presence ofDNP-Glc as donor). In order to assess the relative rates of transfer tothe oxygen and sulfur nucleophiles in the case of Man 2A E429A weestablished a competition reaction in which equimolar amounts of pNP-Xyland its 4-thio analogue 6 were incubated together with DNP-Man as donorand Man2A E429A (FIG. 3). When the concentration of donor was limiting(one third of the concentration of acceptors) we saw exclusive formationof the S-linked disaccharide 10 (a). However, upon addition of furtherdonor to a final concentration twice that of the acceptors the O-linkeddisaccharides (indicated by arrows) were formed (b). Integration of HPLCpeaks indicates that transfer to the thiosugar acceptor is at least100-fold faster than transfer to its oxygen analogue. These results wereconfirmed by ESI-MS analysis.

The glycosynthases have proven to be powerful tools for the synthesis ofO-linked oligosaccharides. In the present invention, the utility of theglycosynthase enzymes is extended and shown to have the further propertyof being able to produce designed thioglycosides in significant yields.Furthermore, the methodology is general, as demonstrated with twodifferent enzymes from two separate glycosidase families, and will proveto be of considerable value in the specific assembly ofthiooligosaccharides for mechanistic and possibly therapeutic use.

A further application of the method of the present invention is theformation of thioglycosidic linkages within proteins in order togenerate thioglycosidic analogues of therapeutic glycoproteins, whichwould be stable to glycosidase-catalysed degradation in vivo. Manytherapeutic proteins, such as Epo, TPA, Enbrel, Ceredase, areglycosylated proteins . . . and the glycosylation is important to theirfunction. Typically these proteins have an oligosaccharide on theirsurface which terminates in a sialyl galactose structure. If all thesialic acids are present the protein has a long circulatory half-life(desirable). However, if any of the sialic acids are missing and thegalactose is exposed the protein gets “taken up” by receptors in theliver and cleared from the circulation. Therefore production ofglycoproteins that do not lose their sialic acids is desirable becausesuch proteins would have much longer circulatory half-lives.Thioglycosidic modification would provide this benefit. However, atpresent it is not possible to chemically assemble thioglycosidic bondsright on the protein surface using conventional approaches, as thereagents are too harsh, and no natural enzymes are available to do thisjob. Mutant thioglycoligase enzymes, however, are perfect for the task.

On a commercial scale, it may be advantageous to immobilize the enzymeto facilitate its removal from a batch of product and subsequent reuse.Such immobilization could be accomplished by use of a fusion protein inwhich the mutant thioligase is engineered onto another protein with highaffinity for an insoluble matrix. For example, a fusion protein with acellulose binding protein prepared in the manner described by Ong etal., “Enzyme Immobilization Using the Cellulose-Binding Domain of aCellulomonas fimi Exoglucanase”, Biotechnology 7: 604-607 (1989) couldbe used in accordance with the invention. Polyhistidine tags may also beused.

The invention will now be further described with reference to thefollowing non-limiting examples:

General Methods:

¹H and ¹³C NMR spectra were recorded on Bruker Avance-300 or Avance-400Spectrometers. Chemical shifts are reported in δ units (ppm) usingresidual ¹H and ¹³C signals of the deuterated solvents as reference:δ_(H) (CDCl₃) 7.26, δ_(H) (CD₃OD) 3.31, δ_(C) (CDCl₃) 77.0, δ_(C)(CD₃OD) 49.0. Electrospray mass spectra were recorded on a PE Sciex API300 LC/MS/MS instrument by direct injection of the compounds in a 1:1CH₃CN/H₂O solution. Melting points were determined with a Mel-Temp IIapparatus and are not corrected. Silica gel 60 (230-400 mesh) fromSiliCycle was used for column chromatography. The petroleum ether usedfor column chromatography had a boiling point range from 35-60° C.Amberlite IR-120PLUS from Aldrich was transformed into the H⁺-formbefore use. All reagents and solvents were purchased from Aldrich,Fluka, Sigma or Fisher Scientific. Solvents were dried over CaH₂(CH₂Cl₂, pyridine, toluene, acetonitrile), over Mg (methanol) or overmolecular sieves 4 Å (DMF). All reactions were carried out under a drynitrogen atmosphere.

EXAMPLE 1 Chemical Synthesis of Deoxythio Sugar Acceptors p-Nitrophenyl2,3,6-tri-O-benzoyl-β-D-galactopyranoside (1)

Benzoyl chloride (1.50 ml, 1.82 g, 12.9 mmol) was added dropwise to asolution of p-nitrophenyl β-D-galactopyranoside (1.00 g, 3.32 mmol) inDMF (15 ml) and pyridine (15 ml) at −20° C. After stirring for 5 h at−5° C. another 0.30 ml of benzoyl chloride (0.36 g, 2.59 mmol) was addeddropwise, and the solution was stirred for 2 h at −5° C. Water (10 ml)was added and the mixture was concentrated by evaporation in vacuo. Theresidue was dissolved in CH₂Cl₂, and the organic phase was washedsequentially with saturated aqueous NaHCO₃, 1 M HCl and brine, driedover MgSO₄, filtered and concentrated in vacuo. Column chromatography(toluene→4:1 toluene/EtOAc) followed by crystallization from hot tolueneyielded 1 (850 mg, 1.39 mmol, 42%); mp 180-181° C.; ¹H-NMR (400 MHz):δ_(H) (CDCl₃): 8.05 (m, 2 H, Ar), 8.0-7.3 (m, 15 H, 3xBz), 7.06 (m, 2 H,Ar), 6.10 (dd, 1 H, J_(2,3) 10.3 Hz, J_(2,1) 7.9 Hz, H-2), 5.48 (dd, 1H, J_(3,2) 10.3 Hz, J_(3,4) 3.2 Hz, H-3), 5.40 (d, 1 H, J_(1,2) 7.9 Hz,H-1), 4.76 (dd, 1 H, J_(6,6) 11.7 Hz, J_(6,5) 5.1 Hz, H-6), 4.68 (dd, 1H, J_(6,6) 11.7 Hz, J_(6,5) 7.7 Hz, H-6), 4.47 (m, 1 H, H-4), 4.31 (m, 1H, H-5), 2.66 (d, 1 H, J_(OH,4) 4.5 Hz, OH); ¹³C-NMR (75 MHz): δ_(C)(CDCl₃): 166.4, 165.8, 165.3, 161.3, 143.0, 133.7, 133.5, 129.9, 129.7,129.7, 129.3, 129.0, 128.6, 128.6, 128.5, 128.5, 125.6, 116.8, 98.8,73.9, 73.2, 69.0, 67.1, 63.0; ESI-MS: m/z=636.5 [M+Na]⁺ (expected forC₃₃H₂₇NO₁₁Na⁺: m/z=636.2).

p-Nitrophenyl4-S-acetyl-2,3,6-tri-O-benzoyl-4-deoxy-4-thio-β-D-glucopyranoside (2)

Trifluoromethanesulfonic anhydride (0.44 ml, 0.75 g, 2.7 mmol) was addeddropwise to a solution of 1 (813 mg, 1.33 mmol) in 20 ml CH₂Cl₂ and 1.2ml pyridine at 0° C. After 1 h at 0° C., CH₂Cl₂ (50 ml) was added, andthe organic layer was washed with saturated aqueous NaHCO₃, 1M HCl andbrine, dried over MgSO₄, filtered and concentrated in vacuo to give 990mg of a yellowish solid (100%). Potassium thioacetate (460 mg, 4.0 mmol)and HMPA (10 ml) were added, and the suspension was stirred at RT for 1h. A mixture of EtOAc/Et₂O (1:1, 50 ml) was added, and the organic layerwas washed twice with water, with brine, dried over MgSO₄, filtered andconcentrated in vacuo. Column chromatography (19:1→3:1 PE/EtOAc) andcrystallization from hot EtOAc yielded 2 as a white powder (520 mg,59%); mp 249° C. (degradation); ¹H-NMR (300 MHz): δ_(H) (CDCl₃): 8.06(m, 2 H, Ar), 8.02-7.31 (m, 15 H, 3xBz), 7.02 (m, 2 H, Ar), 5.84 (dd, 1H, J_(3,4) 10.8 Hz, J_(3,2) 9.3 Hz, H-3), 5.73 (dd, 1 H, J_(2,3) 9.3 Hz,J_(2,1) 7.6 Hz, H-2), 5.43 (d, 1 H, J_(1,2) 7.6 Hz, H-1), 4.79 (dd, 1 H,J_(6,6) 12.0 Hz, J_(6,5) 2.2 Hz, H-6), 4.54 (dd, 1 H, J_(6,6) 12.0 Hz,J_(6,5) 7.2 Hz, H-6), 4.37 (m, 1 H, H-5), 4.10 (t, 1 H, J_(4,5)=J_(4,3)10.8 Hz, H-4), 2.28 (s, 3 H, Ac); ¹³C-NMR (75 MHz): δ_(C) (CDCl₃):192.7, 165.9, 165.6, 165.0, 161.1, 143.1, 138.8, 133.6, 133.6, 129.9,129.8, 129.7, 129.4, 128.8, 128.6, 128.5, 128.4, 125.6, 116.8, 98.3,73.7, 72.5, 71.2, 63.7, 44.3, 30.8; ESI-MS: m/z=694.0 [M+Na]⁺ (expectedfor C₃₅H₂₉NO₁₁SNa⁺: m/z=694.1).

p-Nitrophenyl 4-deoxy-4-thio-β-D-glucopyranoside (3)

A solution of 2 (230 mg, 0.34 mmol) in 10 ml MeOH containing catalyticamounts of MeONa was stirred for 3 h at RT. The mixture was neutralizedwith Amberlite IR-120PLUS (H⁺-form). After filtration, DTT (280 mg, 1.8mmol) in 2 ml of degassed water was added, and N₂ was bubbled throughthe solution for 5 min. After stirring under N₂ overnight the mixturewas concentrated in vacuo. Column chromatography (3:2toluene/EtOAc→EtOAc) afforded 3 as a white powder (80 mg, 0.25 mmol,74%); ¹H-NMR (400 MHz): δ_(H) (d₄-MeOH): 8.22 (m, 2 H, Ar), 7.23 (m, 2H, Ar), 5.10 (d, 1 H, J_(1,2) 7.6 Hz, H-1), 3.94 (dd, 1 H, J_(6,5) 12.3Hz, J_(6,6) 1.9 Hz, H-6), 3.84 (dd, 1 H, J_(6,6) 12.3 Hz, J_(6,5) 4.8Hz, H-6), 3.62 (m, 1 H, H-5), 3.49 (dd, 1 H, J_(2,3) 9 Hz, J_(2,1) 7.6,H-2), 3.41 (dd, 1 H, J_(3,4) 10.2 Hz, J_(3,2) 9.0 Hz, H-3), 2.84 (t, 1H, J_(4,5)=J_(4,3) 10.2 Hz, H-4); ¹³C-NMR (75 MHz) δ_(C) (d₄-MeOH):163.9, 143.9, 126.6, 117.7, 101.5, 79.8, 78.8, 75.7, 62.9, 43.0; ESI-MS:m/z=340.0 [M+Na]⁺ (expected for C₁₂H₁₅NO₇SNa⁺: m/z=340.1).

p-Nitrophenyl 2,3-di-O-benzoyl-α-L-arabinopyranoside (4)

Benzoyl chloride (1.0 ml, 1.25 g, 8.7 mmol) was added dropwise to asolution of p-nitrophenyl α-L-arabinopyranoside (1.00 g, 3.8 mmol) inDMF (30 ml) and pyridine (10 ml) at −20° C. The mixture was allowed towarm to RT overnight while stirring and worked up as compound 1. Columnchromatography (5:1→2:1 PE/EtOAc) and crystallization from EtOAc/heptaneyielded 4 as a white powder (520 mg, 1.11 mmol, 29%); mp 150-151° C.;¹H-NMR (400 MHz): δ_(H) (CDCl₃): 8.18 (m, 2 H, Ar), 8.13-7.40 (m, 10 H,2xBz), 7.08 (m, 2 H, Ar), 5.80 (dd, 1 H, J_(2,3) 6.4 Hz, J_(2,1) 4.2 Hz,H-2), 5.55 (dd, 1 H, J_(3,2) 6.4 Hz, J_(3,4) 3.3 Hz, H-3), 5.52 (d, 1 H,J_(1,2) 4.2 Hz, H-1), 4.46 (m, 1 H, H-4′), 4.17 (dd, 1 H, J_(5,5) 11.9Hz, J_(5,4) 7.0 Hz, H-5), 3.90 (dd, 1 H, J_(5,5) 11.9 Hz, J_(5,4) 3.4Hz, H-5), 2.40 (d, 1 H, J_(OH,4) 5.3 Hz, OH); ¹³C-NMR (100 MHz): δ_(C)(CDCl₃): 165.9, 165.0, 161.2, 142.9, 133.8, 133.7, 129.9, 129.8, 128.8,128.6, 128.6, 125.8, 116.5, 96.8, 71.4, 69.0, 65.1, 62.9; ESI-MS:m/z=502.0 [M+Na]⁺ (expected for C₂₅H₂₁NO₉Na⁺: m/z=502.1).

p-Nitrophenyl4-S-acetyl-2,3-di-O-benzoyl-4-deoxy-4-thio-β-D-xylopyranoside (5)

The partially protected glycoside 4 (340 mg, 0.71 mmol) was treated asdescribed for the preparation of 2. Column chromatography (6:1→2:1PE/EtOAc) gave xyloside 5 as a white foam (215 mg, 0.4 mmol, 56%);¹H-NMR (400 MHz): δ_(H) (CDCl₃): 8.19 (m, 2 H, Ar), 8.1-7.35 (m, 10 H,2xBz), 7.09 (m, 2 H, Ar), 5.60 (m, 1 H, H-3), 5.57 (m, 1 H, H-2), 5.50(d, 1 H, J_(1,2) 4.9 Hz, H-1), 4.42 (dd, 1 H, J_(5,5) 12.2 Hz, J_(5,4)4.2 Hz, H-5), 4.07 (m, 1 H, H-4), 3.75 (dd, 1 H, J_(5,5) 12.2 Hz,J_(5,4) 7.5 Hz, H-5), 2.35 (s, 3 H, Ac); ¹³C-NMR (100 MHz): δ_(C)(CDCl₃): 193.4, 165.2, 165.0, 161.1, 143.0, 133.6, 129.9, 128.9, 128.8,128.5, 125.8, 116.6, 97.7, 70.2, 69.8, 63.4, 41.3, 30.7; ESI-MS:m/z=560.0 [M+Na]⁺ (expected for C₂₇H₂₃NO₉SNa⁺: m/z=560.1).

p-Nitrophenyl 4-deoxy-4-thio-β-D-xylopyranoside (6)

The protected 4-deoxy-4-thioxyloside 5 (190 mg, 0.35 mmol) wasdeprotected as described for compound 3. Column chromatography (1:1→1:3PE/EtOAc) gave 6 as a white powder (70 mg, 0.24 mmol, 69%); ¹H-NMR (400MHz): δ_(H) (d₄-MeOH): 8.20 (m, 2 H, Ar), 7.18 (m, 2 H, Ar), 5.03 (d, 1H, J_(1,2) 7.6 Hz, H-1), 3.98 (dd, 1 H, J_(5,5) 11.8 Hz, J^(5,4) 5.0 Hz,H-5), 3.52 (t, 1 H, J_(5,5)=J_(5,4) 11.8 Hz, H-5), 3.44 (dd, 1 H,J_(2,3) 8.9 Hz, J_(2,1) 7.6 Hz, H-2), 3.32 (m, 1 H, H-3), 2.84 (m, 1 H,H-4); ¹³C-NMR (100 MHz): δ_(C) (d₄-MeOH): 163.7, 143.9, 126.6, 117.6,102.3, 78.6, 75.9, 69.2, 42.0; ESI-MS: m/z=310.0 [M+Na]⁺ (expected forC₁₁H₁₃NO₆SNa⁺: m/z=310.0).

Enzymatic Synthesis of Thiooligosaccharides

The deoxythio sugars 3 or 6 (20 mM), the DNP-donors 2,4-dinitrophenylβ-D-glucopyranoside (DNP-Glc) or 2,5-dinitrophenyl β-D-mannopyranoside(DNP-Man) (30 mM) and the mutant enzymes Abg E171A or Man2A E429A (˜1 mgml⁻¹) were incubated for ˜3 h at RT in phosphate buffer (80 mM). DNP-Glcor DNP-Man was added to a total concentration of 45 mM, and the solutionwas incubated at RT for ˜1 h. After lyophilization standardper-O-acetylation with pyridine/Ac₂O and subsequent workup wasperformed. The final purification by column chromatography (9:1→1:1toluene/EtOAc) yielded products 7-10.

p-Nitrophenyl(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1→4)-S-2,3,6-tri-O-acetyl-4-deoxy-4-thio-β-D-glucopyranoside(7)

25 mg (64%); mp 161.5-162° C. (hot toluene); ¹H-NMR (400 MHz): δ_(H)(CDCl₃): 8.19 (m, 2 H, Ar), 7.09 (m, 2 H, Ar), 5.29-5.16 (m, 4 H, H-1,H-2, H-3), 5.06 (t, 1 H, J_(4′,3′)=J_(4′,5′)9.8 Hz, H-4′), 4.94 (t, 1 H,J_(2′,1′)=J_(2′,3′)9.6 Hz, H-2′), 4.77 (d, 1 H, J_(1′,2′)10 Hz, H-1′),4.64 (dd, 1 H, J_(6,6) 12.1 Hz, J_(6,5) 1.7 Hz, H-6), 4.38 (dd, 1 H,J_(6,6) 12.1 Hz, J_(6,5) 5.5 Hz, H-6), 4.31 (dd, 1 H, J_(6′,6′)12.4 Hz,J_(6′,5′)2.2 Hz, H-6′), 4.13 (dd, 1 H, J_(6′,6′)12.4 Hz, J_(6′,5′)4.8Hz, H-6′), 4.06 (m, 1 H, H-5), 3.75 (m, 1 H, H-5′), 3.05 (t, 1 H,J_(4,5)=J_(4,3) 10.6 Hz, H-4), 2.10, 2.09, 2.07, 2.06, 2.04, 2.02, 2.00(7xs, 21 H, 7xAc); ¹³C-NMR (100 MHz): δ_(C) (CDCl₃): 170.4, 170.1,170.0, 170.0, 169.3, 169.3, 169.2, 161.3, 143.2, 125.7, 116.7, 98.0,81.7, 75.9, 74.6, 73.6, 72.4, 70.3, 69.9, 68.1, 63.3, 61.9, 45.8,20.7-20.4 (7x); ESI-MS: m/z=796.0 [M+Na]⁺ (expected for C₃₂H₃₉NO₁₉SNa⁺:m/z=796.2).

p-Nitrophenyl(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-(1→4)-S-2,3-di-O-acetyl-4-deoxy-4-thio-βD-xylopyranoside(8)

29 mg (79%); mp 122-123° C. (EtOAc/heptane); ¹H-NMR (400 MHz): δ_(H)(CDCl₃): 8.20 (m, 2 H, Ar), 7.07 (m, 1 H, Ar), 5.21-5.11 (m, 4 H, H-1,H-2, H-3, H-3′), 5.07 (t, 1 H, J_(4′,3′)=J_(4′,5′)9.9 Hz, H-4′), 5.00(dd, 1 H, J_(2′,1′)10 Hz, J_(2′,3′)9.3 Hz, H-2′), 4.63 (d, 1 H,J_(1′,2′)10 Hz, H-1′), 4.29 (dd, 1 H, J_(5′,5′)12.4 Hz, J_(5,4) 4.5 Hz,H-5), 4.19 (d, 2 H, J_(6′,5′)3.5 Hz, 2xH-6′), 3.74 (dt, 1 H,J_(5′,4′)9.9 Hz and J_(5′,6′)3.5 Hz, H-5′), 3.65 (dd, 1 H, J_(5,5) 12.4Hz, J_(5,4) 9.6 Hz, H-5), 3.23 (m, 1 H, H-4), 2.10, 2.06, 2.05, 2.03,2.02, 2.00 (6xs, 18 H, 6xAc); ¹³C-NMR (100 MHz): δ_(C) (CDCl₃): 170.4,170.0, 169.9, 169.2, 169.2, 169.0, 161.1, 143.0, 125.7, 116.5, 98.1,81.9, 76.0 (x2), 73.6, 71.3, 69.9, 67.9, 65.6, 61.8, 42.7, 20.5 (6x);ESI-MS: m/z=724.5 [M+Na]⁺ (expected for C₂₉H₃₅NO₁₇SNa⁺: m/z=724.2).

p-Nitrophenyl(2,3,4,6tetra-O-acetyl-β-D-mannopyranosyl)-(1→1)-S-2,3,6-tri-O-acetyl-4-deoxy-4-thio-β-D-glucopyranoside(9)

5 mg (35%); ¹H-NMR (400 MHz): δ_(H) (CDCl₃): 8.20 (m, 2 H, Ar), 7.09 (m,2 H, Ar), 5.36 (dd, 1 H, J_(2′,3′)3.5 Hz, J_(2′,1′)1 Hz, H-2′),5.32-5.17 (m, 4 H, H-1, H-2, H-3, H-4′), 5.09 (dd, 1 H, J_(3′,4′)10.1Hz, J_(3′,2′)3.5 Hz, H-3′), 4.98 (d, 1 H, J_(1′,2′)1 Hz, H-1′), 4.61(dd, 1 H, J_(6,6) 12.2 Hz, J_(6,5) 2.1 Hz, H-6), 4.46 (dd, 1 H, J_(6,6)12.2 Hz, J_(6,5) 5.4 Hz, H-6), 4.33 (dd, 1 H, J_(6′,6′)12.4 Hz,J_(6′,5′)2.4 Hz, H-6′), 4.15 (dd, 1 H, J_(6′,6′)12.4 Hz, J_(6′,5′)5.5Hz, H-6), 4.08 (m, 1 H, H-5), 3.75 (m, 1 H, H-5), 3.07 (t, 1 H,J_(4,5)=J_(4,3) 10.7 Hz, H-4), 2.20 2.12, 2.10, 2.07, 2.06, 2.06, 1.98(7xs, 21 H, 7xAc); ¹³C-NMR (75 MHz): δ_(C) (CDCl₃): 170.5, 170.3, 170.3,170.0, 169.8, 169.6, 169.2, 161.2, 143.2, 125.7, 116.6, 98.1, 79.3,74.3, 72.1, 71.5, 69.9, 69.7, 65.7, 45.1, 20.6 (7x); ESI-MS: m/z=796.0[M+Na]⁺ (expected for C₃₂H₃₉NO₁₉SNa⁺: m/z=796.2).

p-Nitrophenyl(2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)-(1→4)-S-2,3-di-O-acetyl-4-deoxy-4-thio-β-D-xylopyranoside(10)

25 mg (82%); ¹H-NMR (400 MHz): δ_(H) (CDCl₃): 8.20 (m, 2 H, Ar), 7.07(m, 2 H, Ar), 5.45 (d, 1 H, J_(2′,3′)3.5 Hz, H-2′), 5.26-5.12 (m, 4 H,H-1, H-2, H-3, H-4′), 5.07 (dd, 1 H, J_(3′,4′)10.1 Hz, J_(3′,2′)3.5 Hz,H-3′), 4.87 (s, 1 H, H-1′), 4.28-4.15 (m, 3 H, H-5, H-6′, H-6′), 3.75(m, 1 H, H-5′), 3.66 (dd, 1 H, J_(5,5) 12.4 Hz, J_(5,4) 10.0 Hz, H-5),3.28 (m, 1 H, H-4), 2.20, 2.10, 2.08, 2.06, 2.04, 2.03, 1.97 (6xs, 18 H,6xAc); ¹³C-NMR (75 MHz): δ_(C) (CDCl₃): 170.4, 170.2, 170.0, 169.9,169.6, 169.3, 161.1, 143.1, 125.8, 116.5, 98.3, 79.8, 76.8, 71.7, 71.5,70.9, 69.8, 65.5 (2x), 62.6, 43.0, 20.5 (6x); ESI-MS: m/z=724.5 (M+Na]⁺(expected for C₂₉H₃₅NO₁₇SNa⁺: m/z=724.2).

EXAMPLE 2 Competition Study for Evaluation of Relative Rates

pNP-Xyl (5.5 mM), 6 (5.5 mM), DNP-Man (3.75 mM) and Man2A E429A (˜1 mgml⁻¹) were incubated in phosphate buffer (50 mM, pH 6.8) for 3 h at RT.After removal of an aliquot DNP-Man was added to a final concentrationof 22 mM. After 5 h at RT another aliquot was taken. The aliquots werediluted 1:3 with acetonitrile and centrifuged before applying to theHPLC. HPLC analysis was performed using a Waters 600E multisolventdelivery system with acetonitrile (A)/water (B) as mobile phase (lineargradient: 80% A→60% A in 15 min. flow: 1 ml min⁻¹), a Waters 2486 Dual λAbsorbance Detector (detection at 280 nm), a TOSO HAAS Amide 80 column(4.6×250 mm) and Millenium 3.20 software.

EXAMPLE 3

Kinetic analysis was performed with 2,4-DNP β-D-glucopyranoside (125 mM)as donor and with pNP 4-deoxy-4-thio-β-D-glucopyranoside as acceptor at30° C. in 50 mM NaPi, 145 mM NaCl pH 7.1 (FIG. 7). It has been shownpreviously, that the K_(M)-value of the donor is extremely low (<1 μM),and that no donor substrate inhibition is seen. Enzymatic backgroundhydrolysis of the donor together with acceptor substrate inhibition makeit difficult to exactly determine K′_(m) and k′_(cat), but it can becalculated from the highest observed rate of DNP release, that k′_(cat)is higher than 50 min⁻¹.

EXAMPLE 4

Saturation mutagenesis at the acid/base position (E171) of Abg revealednew functional mutations with functionality as thioligases as follows:Abg E171G, Abg E171Q, E171S, E171T, E171M, E171F, E171L, E171I, E171N.

EXAMPLE 5

An endo acting retaining β-glycosidase from Cellulomonas fimi (Cex) wassuccessfully transformed into a functional thioglycoligase by mutationof the catalytic acid/base residue E127. This is especially interesting,as the conversion of Cex into a glycosynthase showed no success.Functional donors for Cex E127A were DNP β-cellobioside and pNP4′-deoxy-4′-thio-β-cellobioside, the latter resulting in selfcondensation to form the thiolinked tetrasaccharide. Functionalacceptors were 4′-deoxy-4′-thio-cellobiose, pNP4′-deoxy-4′-thio-b-cellobioside and pNPβ-D-4-deoxy-4-thio-glucopyranoside.

EXAMPLE 6

The endo mannanase Man26A from Cellvibrio japonicus has been transformedinto the acid/base mutant E212A. TLC and ESI-MS experiments indicate,that Man26A E212A is a functional thioglycoligase catalyzing thetransglycosylation reaction of DNP mannobioside with pNP4′-deoxy-4′-thio-b-cellobioside:

EXAMPLE 7

Cloning of Abg acid/base mutant: The E171A mutation was introduced usingthe megaprimer method and pET29AbgHis6 (Mayer et al., FEBS Letters, 466,40-44) as template. The primers used were p29fwd:

-   5′ GGG GAC AAG TTT GTA CAA AAA AGC AGG CTA AGA AGG AGA TAT ACA TAT G    (Seq. ID. No. 1) and AbgE171A:

5′ CGC GCA CCA AGG CGC GTT GAA CGT TGC AAC CGC ATC (Seq. ID No. 2) inthe first reaction. For the second reaction the product of the first PCRwas use as megaprimer in combination with p29rev: 5′ GGG GAG GAG TTT GTACAA GAA AGC (Seq. ID. No 3) TGG GTC TGA GTG GTG GTG GTG GTG.The resulting product was inserted into the pDONR201 vector andsubsequently transferred to pDEST14 using the Gateway cloning system(Gateway Cloning Technology Manual Version 1. GibcoBRL LifeTechnologies). Protein expression and purification was performed asdescribed in Mayer et al.(Mayer et al., FEBS Letters, 466, 40-44).

REFERENCES

The following references, all of which are incorporated herein byreference, are cited herein:

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1-19. (canceled)
 20. A mutant form of a glycosidase enzyme, said enzymebeing selected from among glycosidase enzymes having two catalyticallyactive amino acids with carboxylic acid side chains within the activesite of the wild-type enzyme including a catalytically active amino acidacting as an acid, base, or acid/base catalyst, said mutant enzyme beingmutated to replace the catalytically active amino acid acting as anacid, base or acid/base catalyst with a different amino acid having anon-carboxylic acid side chain.
 21. The enzyme of claim 20, wherein thedifferent amino acid has a side chain that is approximately equal insize to or smaller than the smaller chain of the replaced amino acid.22. The enzyme of claim 21, wherein the different amino acid is selectedfrom the group consisting of alanine, glycine, valine, leucine,isoleucine, serine, threonine, cysteine, methionine, asparagine,glutamine, histidine, proline, phenylalanine, and tyrosine.
 23. Theenzyme of claim 20, wherein the mutant enzyme is formed by replacing theamino acid in the active site of an enzyme selected from the groupconsisting of β-glucosidases, β-galactosidases, β-mannosidases,β-N-acetyl glucosaminidases, β-N-acetyl galactosaminidases,β-xylosidases, β-fucosidases, cellulases, xylanases, galactanases,mannanases, hemicellulases, amylases, glucoamylases, α-glucosidases,α-galactosidases, α-mannosidases, α-N-acetyl glucosaminidases,α-N-acetyl galactosaminidases, α-xylosidases, α-fucosidases, andneuraminidases/sialidases.
 24. The enzyme of claim 20, wherein themutant enzyme is a mutant of Agrobacterium β-glucosidase.
 25. The enzymeof claim 24, wherein the mutant enzyme is selected from the groupconsisting of AbgE171A, E171G, E171Q, E171S, E171T, E171M, E171F, E171L,E171I, and E171N.
 26. The enzyme of claim 20, wherein the mutant enzymeis a mutant of an endo-acting retaining β-glycosidase of Cellulomonasfimi.
 27. The enzyme of claim 26, wherein the mutant enzyme is CexE127A.
 28. The enzyme of claim 20, wherein the mutant enzyme is a mutantof an endo-mannanase Man26A of Cellvibrio japonicus.
 29. The enzyme ofclaim 28, wherein the mutant enzyme is Man26A E212A.
 30. A method forsynthesizing a thioglycoside having the structure A-S-B, wherein S issulfur and A and B are each sugar moieties, comprising the steps of: (a)combining a donor molecule A-X, where X is a leaving group, and anacceptor molecule HS-B in a reaction mixture; and (b) enzymaticallycoupling the donor molecule to the acceptor molecule using a mutant formof a glycosidase enzyme, said enzyme being selected from amongglycosidase enzymes having two catalytically active amino acids withcarboxylic acid side chains within the active site of the wild-typeenzyme including a catalytically active amino acid acting as an acid,base, or acid/base catalyst, said mutant enzyme being mutated to replacethe catalytically active amino acid acting as an acid, base or acid/basecatalyst with a different amino acid having a non-carboxylic acid sidechain.
 31. The method of claim 30, wherein the leaving group X isdinitrophenol.
 32. The method of claim 30, wherein the donor is selectedfrom the group consisting of 2,4-dinitrophenyl β-D-glucopyranoside(DNP-Glc); 2,5-dinitrophenyl β-D-mannopyranoside (DNP-Man); DNPβ-cellobioside, pNP 4′-deoxy-4′-thio-β-cellobioside and β-D-glucosylazide.
 33. The method of claim 30, wherein the acceptor is selected fromthe group consisting of para-nitrophenyl4-deoxy-4-thio-β-D-glucopyranoside, para-nitrophenyl4-deoxy-4-thio-β-D-galactopyranoside; methylumbelliferyl4-deoxy-4-thio-β-D-glucopyranoside, 4′-deoxy-4′-thio-cellobiose, pNP4′-deoxy-4′-thio-β-cellobioside, and pNPβ-D-4-deoxy-4-thio-glucopyranoside.
 34. The method of claim 30, whereinthe glycosidase enzyme is a stereochemistry inverting enzyme in whichone of the carboxylic acid side chains in the active site functions asan acid catalyst and the other carboxylic acid side chain functions as abase catalyst, and wherein the amino acid having the carboxylic acidside chain which functions as an acid catalyst is replaced in the mutantenzyme.
 35. The method of claim 30, wherein the glycosidase enzyme is astereochemistry retaining enzyme in which one of the carboxylic acidside chains in the active site functions as an acid/base catalyst andthe other carboxylic acid side chain functions as a nucleophile, andwherein the amino acid having the carboxylic acid side chain whichfunctions as an acid/base catalyst is replaced in the mutant enzyme. 36.The method of claim 30, wherein the mutant enzyme is a mutant ofAgrobacterium β-glucosidase, an endo-acting retaining β-glycosidase ofCellulomonas fimi or an endo-mannanase Man26A of Cellvibrio japonicus.37. A thioglycoside prepared by the method of claim
 30. 38. A fusionprotein comprising (a) a mutant form of a glycosidase enzyme, saidenzyme being selected from among glycosidase enzymes having twocatalytically active amino acids with carboxylic acid side chains withinthe active site of the wild-type enzyme including a catalytically activeamino acid acting as an acid, base, or acid/base catalyst, said mutantenzyme being mutated to replace the catalytically active amino acidacting as an acid, base or acid/base catalyst with a different aminoacid having a non-carboxylic acid side chain, and (b) a binding elementfor immobilization of the fusion protein on a solid support.
 39. Thefusion protein of claim 38, wherein the binding element is thecellulose-binding domain of a Cellulomonas fimi exoglucanase.
 40. Thefusion protein of claim 39, wherein the mutant enzyme is a mutant ofAgrobacterium β-glucosidase, an endo-acting retaining β-glycosidase ofCellulomonas fimi or an endo-mannanase Man26A of Cellvibrio japonicus.41. The fusion protein of claim 38, wherein the mutant enzyme is amutant of Agrobacterium β-glucosidase, an endo-acting retainingβ-glycosidase of Cellulomonas fimi or an endo-mannanase Man26A ofCellvibrio japonicus.